Preparation and characterization of DHA dimeric NPs
Firstly, the DHA2-SS was synthesized through the esterification reaction of DHA with dicarboxylic acid (Fig. S1) . After purification by silica gel column chromatography, DHA2-SS was obtained in high yields (>90%) and the chemical construction has been characterized via proton nuclear magnetic resonance (1H NMR) spectroscopy and a linear ion trap mass spectrometer (LTQ-MS). In 1H NMR spectra, the characteristic peak of 10-hydroxyl group at 2.85 ppm corresponding to DHA disappeared, validating the success of esterification reaction and the reaction site was at the 10-hydroxyl group of DHA (Fig. S2). The peak value corresponding to DHA2-SS at around 737 in mass spectrometry was consistent with the theoretical calculated value (Fig. S3), further confirming the structure of DHA dimer. In order to compare the redox responsiveness of DHA2-SS, we also synthesized another DHA dimer with the same length of carbon chain (DHA2-C6) as control (Fig. S4).
It is reported that the organic dimers could self-assemble into NPs in aqueous solution [25–30]. As anticipated, both kinds of resulting dimers formed spherical nanoparticles (abbreviated as SS NPs and C6 NPs, respectively) through nanoprecipitation method as observed via transmission electron microscopy (TEM) (Fig. 1A and 1B). And SS NPs had an average hydrodynamic diameter of approximately 167.2 nm as determined by dynamic light scattering (DLS), which was similar to those of C6 NPs (181.4 nm) (Fig. 1C). These two kinds of NPs were found to be negative, and the zeta potential values were around -20 mV (Fig. 1D). The drug content of SS and C6 NPs was 90.6% and 91.7%, respectively. In addition, SS and C6 NPs both had robust stability with negligible changes in size and size distribution in one week (Fig. 1E). And these NPs also kept stable in PBS (pH 7.4) containing 10% FBS after 24 h (Fig. 1F), and a slight size increase in the first two hours was largely ascribed to the protein absorption on the surface of NPs.
Cellular uptake and in vitro cytotoxicity of DHA dimeric NPs
Human hepatoma HepG2 cells were used to study the cellular internalization of these NPs via confocal laser scanning microscopy (CLSM). The fluorescent dye nile red (NR) was utilized as a marker and encapsulated into the NPs by coassembly with DHA dimer, and the blue fluorescence of Hoechst 33258 was employed to localize the cell nucleus. As exhibited in Fig. 3A, the red fluorescence principally distributed in the cytoplasm of tumor cells in a time-dependent manner (Fig. S5), implying the efficient and sustained cellular uptake. Furthermore, the effect of temperature on cellular uptake has also been carried out. It can be observed that a strong fluorescence signal at 37°C and significantly reduced cellular uptake at 4°C from Fig. S6, suggesting an energy-dependent endocytosis.
Next, the cytotoxicity of DHA dimeric NPs was evaluated against human HepG2 and HeLa cells through a standard MTT assay. As shown in Fig. 3B, both free DHA and SS NPs showed efficient suppression of HepG2 viability in concentration dependent manner for 48 h, and SS NPs exhibited higher cytotoxicity than free DHA. The cell viability of SS NPs was less than 30%, while it was approximately 40% for free DHA at the equivalent DHA concentration. This result may be attributed to the enhanced cellular uptake of NPs into tumor cells and the rapid release of DHA. And SS NPs could quickly release active DHA once in living cells because of the responsiveness of disulfide bond in redox environment. However, C6 NPs exhibited apparent lower cytotoxicity compared with SS NPs (Fig. S7). Similarly, SS NPs still possessed the strongest cellular toxicity towards HeLa cells (Fig. S8). In addition, we evaluated the cytotoxicity of SS NPs toward normal hepatocytes (HL-7702), and also compared its toxicity on HepG2, HeLa and HL-7702 cells. As revealed in Fig. 3C, SS NPs exhibited no significant cellular toxicity against HL-7702 cells, and enhanced cytotoxicity against these two kinds of tumor cells (Fig. S9), indicating the selectivity of DHA2-SS prodrug towards tumor cells. This result is ascribed to the different redox conditions in normal cells and tumor cells.
To further understand the contribution of SS NPs on apoptosis, we stained HepG2 cells with Annexin V-FITC and PI, and analyzed them by flow cytometry. As presented in Fig. 3D, the ratio of early apoptotic cells was 9.40%, 15.00%, and 24.56%, respectively, as the drug concentration increases (20, 40 and 60 µM), validating the cell apoptosis in a concentration-dependent manner. Additionally, we also examined the nuclear morphological changes by CLSM. The cell nucleus emerged as a homogeneous blue chromatin with an organized structure in normal cells, whereas the cells incubated with SS NPs displayed representative morphological changes (Fig. S10), including intense fluorescent spots, nuclear pyknosis, and extensive blebbing, further verifying the apoptosis of tumor cells.
Antitumor mechanism of SS NPs
To elucidate the mechanism of SS NPs in inhibiting tumor cell proliferation and inducing apoptosis, RNA sequencing (RNA-seq) technology was applied to collect the gene expression . The total RNA extracted from SS NPs treatment group (SS) and the control group (C) have been analyzed for quality and integrity by utilizing formaldehyde agarose gel electrophoresis. The results verified that the obtained RNA was intact, undegraded, and suitable for RNA-seq analysis. Meanwhile, principal component analysis (PCA) of samples was performed on the complete dataset , which can display changes of overall gene expression. As shown in the PCA results (Fig. S11), there were two clusters, verifying a distinct directionality between SS NPs treatment and the control groups based on the similarity of gene expression. Subsequently, the differentially expressed genes (DEGs) induced by SS NPs have been identified and described by volcano plots and heatmaps. After comparing with the untreated control group (log2 fold-change ≥ 2.0 and adjusted P value < 0.05), we distinguished 6546 DEGs, including 3288 up-regulated and 3258 down-regulated expression genes (Fig. 4A and 4B). The above results imply that SS NPs play an important role on gene expression of the treated tumor cells.
According to the RNA-seq data, we performed the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for 6546 differentially expressed genes. Three domains were involved: cellular components (CC), biological processes (BP) and molecular function (MF). In the BP domain, a large proportion of genes were associated with cellular process, metabolic process, biological regulation (Fig. 5A). For the CC domain, the enriched genes covered cell, cell part, organelle, membrane and so forth (Fig. 5B). Binding, catalytic activity, transcription regulator activity and molecular function regulator were primarily influenced for the MF domain (Fig. 5C).
On the basis of the DEGs results, we also executed KEGG functional enrichment analysis and pathway classification. The KEGG functional enrichment analysis result (Fig. 5D) demonstrated that multiple pathways have been affected by DHA2-SS NPs, and the signaling pathways of carbon metabolism, biosynthesis of amino acids, fatty acid metabolism, HIF-1 signaling pathway and pathways in cancer were among the top 20 pathways. On the other hand, KEGG pathway classification results (Fig. S12) displayed that there were six branches for KEGG pathways, including cellular processes, environmental information processing, genetic information processing, human diseases, metabolism, and organismal systems. And the DEGs in HepG2 cells after SS NPs treatment were mainly enriched in PI3K-Akt signaling pathway, MAPK signaling pathway and pathways in cancer.
From GO and pathway analysis results, we discovered that the metabolic process was involved in the inhibition of tumor cells proliferation, thus we carried out network statistical analysis on the protein-protein interaction (PPI) of these genes correlated with metabolism. As shown in Fig. 6, the metabolism of carbohydrates, amino acids and lipids have been remarkably regulated in the cells after SS NPs treatment, indicating that SS NPs could induce metabolic reprogramming in tumor cells. Therefore, we concentrated on the metabolism reprogramming of tumor cells after treated with SS NPs.
Apoptosis is a highly regulated process of cell death, which is important to maintain the inherent stability of multicellular organisms, and involves a variety of signal pathways [52–54]. The Bcl-2 protein family contains pro-apoptotic and anti-apoptotic regulators of programmed cell death/apoptosis, and plays a dominant role in regulating cell apoptosis. In this family, Bax gene, a pro-apoptotic member, can form heterodimers with Bcl-2 protein, and the ratio of Bax/Bcl-2 could determine the sensitivity of cells to apoptosis. The activation of Bax can release cytochrome c (Cyt C), and Cyt C activates caspase-9 and downstream caspase-3 through a cascade reaction to promote cell apoptosis, whereas the anti-apoptotic Bcl-2 operates in the opposite way. Therefore, we detected expression of Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9 and Cyt C by western blotting. As displayed in Fig. 7, the expression levels of Bax, caspase-3, caspase-9, and Cyt C were obviously increased, and the expression levels of Bcl-2 was reduced compared to the control group. The above results demonstrate that mitochondrial apoptosis pathway is involved in the apoptosis of HepG2 cells induced by SS NPs.
As we known, tumor cells generally reveal aberrant metabolism due to metabolic reprogramming . As a hallmark of tumor cells, the Warburg effect means that tumor cells rely heavily on glycolysis for energy, rather than oxygen [56–58]. To investigate whether SS NPs treatment could suppress the glycolysis of tumor cells, the glucose uptake, contents of lactic acid and ATP products of HepG2 cells treated with SS NPs were detected. As shown in Fig. 8, the glucose uptake was decreased, and the contents of intracellular ATP and extracellular lactic acid were also declined, indicating that SS NPs may inhibit glycolysis of tumor cells.
Cancer metabolic reprogramming is regulated by multiple pathways, which includes the PI3K-AKT signaling pathway . The activated PI3K-AKT can promote the transition to aerobic glycolysis, and AKT could result in the phosphorylation of some important downstream targets, such as Bcl-2 apoptosis-related family, and mammalian target of rapamycin (mTOR), to protect cells from apoptosis. Meanwhile, this pathway also could regulate HIF-1α through mTOR, and activated HIF-1α is related to the up-regulation of glucose transporters (Gluts) and glycolytic enzymes. To understand whether the activation of PI3K/AKT and HIF-1α were involved in metabolic reprogramming of tumor cells treated by SS NPs, we detected the related protein expression. As displayed in Fig. 9, SS NPs treatment could decrease the expression of glycolytic enzymes, such as PFKP, HK2, LDH, Glut1. The ratio of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR and HIF-1α were also reduced accordingly. Collectively, these findings confirm that SS NPs could induce apoptosis and suppress glycolysis by regulating the PI3K/AKT/HIF-1α signaling pathway.
In vitro antitumor efficacy of DHA dimeric NPs
We further evaluated the anti-cancer activity of SS NPs on H22 tumor-bearing Kunming mice. Mice bearing the tumors were randomly divided into 4 groups with different treatments: PBS, free DHA, C6 NPs, and SS NPs, and injected intravenously at equivalent DHA doses every second day. As illustrated in Fig. 10A, DHA treatment exhibited a moderate inhibitory effect on tumor growth compared with the control group, which is mainly owing to its intrinsic toxicity. Notably, SS NPs exhibited evident antitumor activity, which is more potent than free DHA group. The improved therapeutic efficacy of SS NPs should be ascribed to the multiple advantages of nanoparticle formulations, including enhanced tumor accumulation, effective endocytosis, and rapid drug release in tumor sites. Unsurprisingly, C6 NPs group displayed the weakest tumor growth inhibition effect in these treatment groups on account of the insensitivity of C6 linker to redox microenvironment. What’s more, the tumor weight (Fig. 10B) and the photographs of resected tumors (Fig. 10C) visually demonstrated the greatest tumor inhibition efficacy obtained by SS NPs, further validating the enhanced antitumor effect of disulfide-bond bridged prodrug nanoparticles. In addition, all mice had no significant weight fluctuation during the whole treatment period (Fig. 10D), and there was also no detectable histological damage observed after SS NPs treatment from the hematoxylin and eosin (H&E) stained tissue sections of major organs (heart, liver, spleen, lung, and kidney) (Fig. S13). The above results validate SS NPs at current doses possess favorable biosafety and ignorable systemic toxicity. The H&E staining of tumor slices revealed that SS NPs group exhibited the most severe cellular damage compared with the control group. The tumor cells after SS NPs treatment shrank largely and the tumor tissue significantly decreased. (Fig. 10E). All of the results substantiate that DHA2-SS NPs could be safely used for the in vivo treatment, and possess better treatment effects in contrast with free DHA.